COMPARISON OF DNA METHYLATION PATTERNS AMONG MOUSE-CELL LINES BY RESTRICTION LANDMARK GENOMIC SCANNING

Restriction landmark genomic scanning (RLGS) is a novel method which e nables us to simultaneously visualize a large number of loci as two-di mensional gel spots. By this method, the status of DNA methylation can efficiently be determined by monitoring the appearance or disappearan ce of spots by using a methylation sensitive restriction enzyme. In th e present study, using RLGS with NotI, we examined, in comparison with a brain RLGS profile, the status of DNA methylation of more than 900 loci among three types of mouse cell lines: the embryonal carcinoma ce ll line P19, the stable mesenchymal cell line 10T1/2, and our establis hed neuroepithelial (EM) cell lines. We found that the relative number s of RLGS spots which appeared were less than 3.3% of those surveyed i n all cell lines examined. However, 5 to 14% of spots disappeared, the numbers increasing with an increase in the length of the culture peri od, and many spots: were commonly lost in 10T1/2 and in three EM cell lines. Thus, for these cell lines, many more spots disappeared than ap peared. However, the numbers of spots disappearing and appearing were well balanced, and the ratio in P19 cells was almost equal to that in liver cells in vivo. These RLGS experimental observations suggested th at permanent cell lines such as 10T1/2 are hypermethylated and that ou r newly established EM cell lines are also becoming heavily methylated at common loci. On the other hand, methylation and demethylation seem to be balanced in P19 cells in a manner similar to that in in vivo li ver tissue.